Several tumor-associated antigens (TAA) constitutively expressed by transformed cells of different histotype have been recently identified (Renkvist N. et al. Cancer Immunol. Immunother. 50:3-15,2001).
A number of these TAA can provide multiple immunodominant antigenic peptides recognized by CD8+ cytotoxic T lymphocytes (CTL) in the context of specific HLA class I allospecificities (Renkvist N. et al. Cancer Immunol. Immunother. 50:3-15,2001); furthermore selected TAA, such as for example MAGE (Jager E. et al., J. Exp. Med., 187: 265-270, 1998), NY-ESO-1 (Jager E. et al., J. Exp. Med., 187: 265-270, 1998), SSX (Tureci O, et al. Cancer Res; 56(20):4766-72 1996), tyrosinase (Topalian S. L. et al., J. Exp. Med., 183: 1965-1971, 1996.), Melan-A/MART-1 (Zarour H. M. et al., Proc. Natl. Acad. Sci. USA, 97: 400-405, 2000) concomitantly include epitopes recognized by CD4+ T lymphocytes in the context of specific HLA class II allospecificities, thus being able to induce a TAA-directed humoral immune response (Wang R. F., Trends Immunol., 22: 269-276, 2001).
Different classes of TAA that may play a major role as therapeutic targets have been identified:                i) cancer-testis antigens (CTA), expressed in tumors of various histology but not in normal tissues, other than testis and placenta such as for example MAGE, GAGE, SSX SART-1, BAGE, NY-ESO-1, XAGE-1, TRAG-3 and SAGE, some of which represent multiple families (Traversari C., Minerva Biotech., 11: 243-253, 1999);        ii) differentiation-specific antigens, expressed in normal and neoplastic melanocytes, such as for example tyrosinase, Melan-A/MART-1, gp100/Pmel17, TRP-1/gp75, TRP-2 (Traversari C., Minerva Biotech, 11: 243-253, 1999);        iii) antigens over-expressed in malignant tissues of different histology but also present in their benign counterpart, for example PRAME (Ikeda H. et al., Immunity, 6: 199-208, 1997), HER-2/neu (Traversari C., Minerva Biotech, 11: 243-253, 1999), CEA, MUC-1(Monges G. M. et al., Am. J. Clin. Pathol., 112: 635-640, 1999), alpha-fetoprotein (Meng W. S. et al., Mol. Immunol., 37. 943-950, 2001);        iv) antigens derived from point mutations of genes encoding ubiquitously expressed proteins, such as MUM- 1, β-catenin, HLA-A2, CDK4, and caspase 8 (Traversari C., Minerva Biotech., 11:243-253, 1999);        v) viral antigens (Traversari C., Minerva Biotech., 11: 243-253, 1999).        
In addition to TAA, the cellular elements that are crucial for their effective immunogenicity and efficient recognition by host's T lymphocytes include HLA class I and HLA class II antigens, and co-stimulatory/accessory molecules (e.g., CD40, CD54, CD58, CD80, CD81) (Fleuren G. J. et al., Immunol. Rev., 145: 91-122, 1995).
Among known classes of TAA, CTA are particularly suitable therapeutic targets for active specific immunotherapy of cancer patients, because of their limited expression in normal tissues and their known in vivo immunogenicity in living subjects, in particular mammals, humans included (Jager E. et al., J. Exp. Med., 187: 265-270, 1998; Rejnolds S. R. et al., Int. J. Cancer, 72: 972-976, 1997). However, the heterogeneous expression of specific CTA among neoplastic lesions of different patients limits their biological eligibility to CTA-directed therapeutic vaccination. In fact, malignant lesions of distinct cancer patients can frequently express only selected CTA (Sahin U. et al., Clin. Cancer Res., 6: 3916-3922, 2000), additionally down-regulated (Leth{hacek over (z)} B. et al., Melanoma Res., 7: S83-S88, 1997) and/or heterogeneous (dos Santos N. R. et al., Cancer Res., 60: 1654-1662, 2000) expression of specific CTA within individual neoplastic lesions has also been reported (Jungbluth A. A. et al., Br. J. Cancer, 83: 493-497, 2000). These events, that can occur in vivo separately or concomitantly, may also contribute to the constitutively poor immunogenicity of malignant cells favouring disease progression (Speiser D. E. et al., J. Exp. Med., 186: 645-653, 1997), and may as well lead to in vivo immunoselection of neoplastic cells with the emergence of CTA-negative clones, in the course of immunologic treatment against specific CTA. Thus, immunotherapeutic approaches that focus on the immunologic targeting of distinct immunogenic epitopes of single CTA cannot be applied to large numbers of cancer patients, due to the absence or the possibly down-regulated expression of target CTA in their neoplastic lesions; furthermore, the immunological targeting of single CTA in vivo may generate CTA-loss tumor variants that efficiently escape treatment-induced/amplified CTA-specific immune response. An additional limit to therapeutic approaches that target single CTA derive from their heterogeneous intralesional expression (Schultz-Thater E. et al., Br. J. Cancer, 83: 204-208, 2000), moreover, the presentation of distinct immunogenic epitopes of single CTA by specific HLA class I or HLA class II allospecificities allows treatment only of patients with certain defined HLA phenotypes.
To partially obviate to these limitations, recent therapeutic strategies are utilizing more than one immunogenic epitope of single or multiple CTA, or the whole CTA protein as vaccinating agent (Conference on Cancer Vaccines, Eds. Ferrantini M. and Belardelli F., Rome-Italy, Nov. 15-16, 1999; http://www.cancerresearch.org).
Accordingly, there is a strongly felt need for a cancer vaccine which can overcome the drawbacks of the state of the art, in particular poor immunogenicity, in vivo immunoselection, the possibility to practice a cancer vaccine on a wide population of cancer patients, not limited to the specific single targeted CTA, or TAA, and in that the cancer vaccine not be “restricted” to selected HLA class I and/or HLA class II antigens.
Recent in vitro evidences have demonstrated that the expression of all CTA genes that have been investigated, among the so far known, is induced or up-regulated in neoplastic cells of different histology following their exposure to DNA hypomethylating agents (dos Santos N. R. et al., Cancer Res., 60: 1654-1662, 2000; Weber J. et al., Cancer Res., 54: 1766-1771, 1994) CTA induction was found to be persistent being still detectable several weeks after the end of treatment. These findings support the notion that CTA belong to a class of TAA that is comprehensively regulated by DNA methylation. Furthermore, treatment of neoplastic cells with DNA hypomethylating agents induced a concomitant and persistent up-regulation of their expression of HLA class I antigens and of investigated HLA class I allospecificities, and also up-modulated the expression of the co-stimulatory/accessory molecules CD54 and CD58 (Coral S. et al., J. Immunother., 22: 16-24, 1999).
Notwithstanding their promising therapeutic profile, CTA, however, show a number of drawbacks, such as that specific CTA so far investigated show a heterogeneous expression within distinct neoplastic lesions, with the co-existence of CTA-positive and -negative malignant cells; that only selected CTA among the ones so far identified may be expressed on distinct neoplastic lesions, independently from their hystological origin; that threshold levels of expression of specific CTA on neoplastic cells are required for their recognition by CTA-specific CTL and that vaccination against a specific CTA requires an appropriate HLA class I and, for selected CTA also HLA class II phenotype of patients.
Due to their unique biologic features, selected CTA are being utilized in different clinical trials that aim to induce or potentiate a CTA-specific immune response in patients affected by malignant diseases of different histology. Diverse strategies are currently utilized for the in vivo administration of therapeutic CTA in the clinic or for the generation of more powerful vaccinating tools at pre-clinical level (dos Santos N. R. et al., Cancer Res., 60: 1654-1662, 2000; Weber J. et al., Cancer Res., 54: 1766-1771, 1994) as the person expert in the art is aware of. Noteworthy, mainly due to a number of technical and practical limitations, only a limited number of immunogenic epitopes of specific CTA, or single whole CTA protein are currently utilized in the clinic for the therapeutic purposes. Following is a list including the main strategies already utilized, or hypothesised so far, to administer CTA to cancer patients; it should also be emphasised that identical strategies are utilized to administer to patients TAA that belong to the other classes of so far known TAA, and that different adjuvants and/or carriers are sometimes utilized to potentiate the immunogenicity of therapeutic agents.                Synthetic peptides representing immunogenic epitope(s) of single or multiple CTA recognized by CD8+ T cells (Conference on Cancer Vaccines, Eds. Ferrantini M. and Belardelli F., Rome-Italy, Nov. 15-16, 1999.        Liposome-encapsulated synthetic peptides representing immunogenic epitope(s) of single or multiple CTA (Steller M. A. et al., Clin. Cancer Res., 4: 2103-2109, 1998).        Whole synthetic protein of a single CTA (Conference on Cancer Vaccines, Eds. Ferrantini M. and Belardelli F., Rome-Italy, Nov. 15-16, 1999.        Recombinant viral vectors expressing epitopes of single or multiple CTA recognized by CD8+ T cells (Jenne L. et al., Trends Immunol., 22:102-107, 2001).        Naked DNA shooting (Park J. H. et al., Mol. Cells, 9: 384-391, 1999).        Autologous PBMC/macrophages loaded ex vivo with synthetic peptides representing epitopes of single or multiple CTA recognized by CD8+ T cells (Conference on Cancer Vaccines, Eds. Ferrantini M. and Belardelli F., Rome-Italy, Nov. 15-16, 1999).        Autologous dendritic cells loaded ex vivo with synthetic peptides representing epitopes of single or multiple CTA recognised by CD8+ T cells or loaded with whole synthetic protein of a single CTA, or loaded with whole tumour cell preparations (Conference on Cancer Vaccines, Eds. Ferrantini M. and Belardelli F., Rome-Italy, Nov. 15-16, 1999 Jenne L. et al., Trends Immunol., 22:102-107, 2001).        Autologous dendritic cells transfected or transduced ex vivo with DNA/RNA to express full-length CTA or fused with whole tumor cells (Jenne L. et al., Trends Immunol., 22:102-107, 2001);        Autologous T lymphocytes transfected or transduced ex vivo with DNA/RNA to express full-length CTA.        
As far as autologous cancer vaccines, which the present invention refers to as the main object, a number of patent references may be cited. WO 99/42128 discloses methods for determining the HLA transcription or expression profile of a solid tumor, for selection of appropriate treatments and/or for monitoring progress of the tumor. The purpose of this reference is to inhibit some isoforms of HLA-G in order to increase the native antitumor response. The method comprises extracting cells from a tumor sample, lysing them and reacting the lysate with antibodies directed against HLA Class I antigens.
DE 29913522 provides an apparatus for preparing a cancer vaccine by submitting tumor cells extracted from a patient to pressures of 200-9000 bar, in order to kill or damage the cells while leaving their surface intact then reinjecting the cells to the patient.
WO 00/02581 discloses a telomerase protein or peptide, capable of inducing a T cell response against an oncogene or mutant tumor suppressor protein or peptide. Said peptides are used for a cancer vaccine.
WO 00/18933 discloses DNA constructs causing expression of functionally inactive, altered antigens which are unaltered with respect to the efficiency of transcription and translation of DNA, RNA or the generation of antigenic peptides. The patient affected by cancer is treated by the administration of the RNA or plasmid DNA encoding an altered human cancer associated antigen, in particular PSMA antigen. In a different embodiment, autologous dendritic cells that have been exposed in vitro to the RNA or the plasmid DNA are used as vaccine.
WO 00/20581 discloses a cancer vaccine comprising a new isolated MAGE-A3 human leukocyte antigen (HLA) class II-binding peptide. The peptide can also be used to enrich selectively a population of T lymphocytes with CD4+ T lymphocytes specific the said peptide. Said enriched lymphocytes are also used as cancer vaccine.
WO 00/25813 discloses universal Tumor-Associated Antigen (TAA) binding to a major histocompatibility complex molecule. The method of treatment comprises administering a nucleic acid molecule encoding the TAA, which is processed by an antigen-presenting cell which activates cytotoxic lymphocytes and kills cells expressing TAA. Other than the specific hTERT peptide, the identification of different TAAs is enabled by a complex computer-aided method synthesis of the computer-designed peptide and biological assays for confirmation of the usefulness of the peptide.
WO 00/26249 discloses fragments of human WT-1 protein or human gata-1 protein. These peptide fragments are used for cancer vaccine through activation of cytotoxic T lymphocytes (CTL).
U.S. Pat. No. 6,077,519 provides a cancer vaccine comprising a composition of T cell epitopes recovered through acid elution of epitopes from tumor tissue.
WO 00/46352 provides a cancer vaccine comprising human T lymphocytes that express a functional CD86 molecule. T lymphocytes are obtained by subjecting T cells to at least two sequential stimuli, each involving at least one activator (an antibody anti CD2, 3 or 28) and a cytokine (interleukine) that stimulates T cell proliferation.
Coral S. et al. Journal of Immunotherapy 22(1):16-24, 1999, teach that the immunogenic potential of melanoma cells and their recognition by the host's cytotoxic cells depend on the presence and on the level of expression of Human Leukocytic Antigen (HLA) class I antigens, costimulatory molecules and melanoma-associated antigens (MAA) on neoplastic cells. There may be a suggestion that 5-AZA-CdR for use in active and/or passive specific immunotherapy for human melanoma through its systemic administration might enhance melanoma cells recognition by cytotoxic cells.
Momparler, Anticancer Drugs Apr; 8(4):358-68, 1997, mentions 5-AZA-CdR as chemotherapic.
Shichijo S. et al Jpn. J. Cancer Res. 87, 751-756, July 1996, investigated whether the demethylating agent 5-AZA-CDR induces MAGE 1, 2, 3 and 6 in normal and malignant lymphoid cells in order to better understand the mechanisms of their expression in the cells. The authors showed the induction of investigated CTA in selected samples tested and discussed that demethylation is not a sufficient stimulus to induce MAGE genes in all cases and that their results should lead to a better understanding of mechanisms of MAGE genes expression in cells. No perspective therapeutic implications were suggested.